Truncation signal losses with FFPE sections

As discussed above, commercial probes for deletion detection from competitors use two color FISH, which included the gene of interest and a centromere specific reference, probe from the same chromosome (Figure 2). The major limitation of simple two color FISH in a widely spaced configuration (centromere and gene of interest) is that detecting copy number changes in tissue sections will lead to higher false positive rates for deletions as nuclei are scored. The distance between the PTEN gene and the centromere control probes (as used by current competitors) means that their distribution in the nuclear three-dimensional space is essentially random. Thus the chances of the control probe being excluded from the nucleus during the sectioning process are just as high as for the gene of interest. This "truncation effect" occurs because part of the cell and nucleus can be lost during the sectioning process, leading to loss of signal from the nucleus being scored. This problem is not a major consideration for the detection of amplifications or gain, where extra copies or signal clusters will still be clearly visible (REF). However, if the target chromosomal change is a deletion then the cutoff level for truncation signal losses (i.e. the false positive rate) can be quite high (see Figure 4).

FIGURE 4 Three dimensional schematic depiction of commercial (competitor) two color interphase FISH using normal cells that will have two red and two green spots per nucleus (see Figure 2 for details of probes). Because of the large distance and random distribution of green and red spots in the nucleus the truncation effect will have an equal probability of leading to signal loss of both probes, so that the control green centromeric probe is not as informative in discriminating between real chromosomal deletion events and false truncation exclusions by sectioning.

The expected proportion of nuclei exhibiting truncation losses when using two color FISH has to be carefully determined using appropriate negative controls. The influence of both diameter and shape of nuclei is an important consideration when evaluation truncation false positive levels with interphase FISH. Nuclei in sections typically have a mean diameter of 8-10 microns, and confocal analysis has shown that intact nuclei often have an elliptical shape rather than the perfect spheres shown schematically in figures 4 and 5. In a recent study of truncation loss of signal by interphase FISH (in 5 micron histological sections of normal cells) was found to have threshold levels of only ~ 20% or normal bone marrow nuclei exhibiting signal losses due to truncations. In contrast ~60% of nuclei in normal liver sections exhibit truncation losses (Wilkins et al.). This variation in false positive rates for detecting losses is due to the cutting artifacts of truncation, that have a greater affect when the larger and irregularly shaped liver cells are more frequently bisected than bone marrow cells, using the 5 micron sections. It is therefore essential that the false positive, likely to come from truncation, be determined by comparison with normal nuclei to the neoplastic tissue nuclei of interest, for all deletion FISH assays. One practical solution for minimizing the truncation effects is the use of 3 or 4 color FISH and proper probe design in the region subject to deletion (i.e. the genomic distances between control probes and the tumor suppressor gene of interest.) The false-positive rate due to truncation can be reduced by choosing an optimal genomic distance between each of the probes that constitutes the three color FISH assay design. In this way it is possible to determine whether a given gene is deleted or not with a higher degree of certainty. We compare 4 color FISH (DDP in house probe set) to 2 color FISH (competitor) is presented in Table 1 below.

FIGURE 5 Principle of DDP in house FISH assay design. LEFT PANEL: Position of probes used by our group to detect PTEN deletion events using four color FISH in prostate cancer (REFS). RIGHT PANEL: Three dimensional schematic depictions of four color interphase FISH using normal cells that will be expected to have two red, green, blue and violet spots per nucleus. The blue centromere probe is used to determine if monotony 10 may be present. The DDP three probe set comprise a red PTEN probe which is flanked by the violet TSPAN15 on the centromeric side, and by the green FAS gene on the telomeric side. Because of the close proximity or flanking control probes, the truncation effect is more likely to impact the entire DDP set so that scoring algorithms for deletion can be designed with improved discrimination between real deletion events and the false truncation exclusions caused by sectioning. In the DDP assay analyses of normal nuclei such as in the schematic section shown here are used to determine threshold levels of "technical loss events" for each probe due truncation exclusion during sectioning.